The members of the genus Alphavirus are positive-sense RNA viruses, which are predominantly transmitted to vertebrates by a mosquito vector. Alphavirus disease in humans can be severely debilitating, and depending on the particular viral species, infection may result in encephalitis and possibly death. In recent years, alphaviruses have received significant attention from public health authorities as a consequence of the dramatic emergence of chikungunya virus in the Indian Ocean islands and the Caribbean. Currently, no safe, approved or effective vaccine or antiviral intervention exists for human alphavirus infection. The molecular biology of alphavirus RNA synthesis has been well studied in a few species of the genus and represents a general target for antiviral drug development. This review describes what is currently understood about the regulation of alphavirus RNA synthesis, the roles of the viral non-structural proteins in this process and the functions of cis-acting RNA elements in replication, and points to open questions within the field.

Summary The mechanisms utilized by viruses to protect their transcripts from the cellular RNA decay machinery, as well as the biological relevance of this protection, are largely unknown. We demonstrate that Sindbis virus uses U-rich 3’ UTR sequences in its RNAs to recruit the cellular HuR protein during infections of both human and mosquito cells. HuR binds viral RNAs with high specificity and affinity. Furthermore, Sindbis virus induces the selective movement of HuR protein out of the nucleus of mammalian cells during infection thereby increasing the cytoplasmic pool of the protein available to the virus. Finally, knockdown of HuR results in a significant increase in the rate of decay of Sindbis virus RNAs and diminishes viral yields in both human and mosquito cells. Collectively these data indicate that Sindbis virus, and likely other alphaviruses, usurp the HuR protein to avoid the cellular mRNA decay machinery and maintain a highly productive infection.

Alphaviruses are enveloped positive-sense RNA viruses that exhibit a wide host range consisting of vertebrate and invertebrate species. Previously we have reported that the infectivity of Sindbis virus (SINV), the model alphavirus, was largely a function of the cell line producing the viral particles. Mammalian-cell-derived SINV particles, on average, exhibit a higher particle-to-PFU ratio than mosquito cell-derived SINV particles. Nevertheless, the outcome of nonproductive infection, the molecular traits that determine particle infectivity and the biological importance of noninfectious particles were, prior to this study, unknown. Here, we report that the incoming genomic RNAs of noninfectious SINV particles undergo rapid degradation following infection. Moreover, these studies have led to the identification of the absence of the 5= cap structure as a primary molecular determinant of particle infectivity. We show that the genomic RNAs of alphaviruses are not universally 5= capped, with a significant number of noncapped genomic RNA produced early in infection. The production of noncapped viral genomic RNAs is important to the establishment and maintenance of alphaviral infection. IMPORTANCEThis report is of importance to the field of virology for three reasons. First, these studies demonstrate that noncapped Sindbis virus particles are produced as a result of viral RNA synthesis. Second, this report is, to our knowledge, the first instance of the direct measurement of the half-life of an incoming genomic RNA from a positive-sense RNA virus. Third, these studies indicate that alphaviral infection is likely a concerted effort of infectious and noninfectious viral particles.A lphaviruses are enveloped, positive-sense RNA viruses that are cyclically transmitted between sylvatic vertebrate reservoir hosts and a mosquito vector during the enzootic cycle. The maintenance of this cyclical transmission is vital to viral fitness, as prolonged serial passage within a single host results in attenuation in the alternate host (1-4). Moreover, the ultimate outcome of alphaviral infection differs between vertebrate and invertebrate hosts, as infection of a vertebrate host results in acute cytolytic infection whereas infection of invertebrate hosts often results in persistent infection with minimal cell death (5-11). The widespread geographic distribution of competent vector mosquito species leading to contact with immunologically naive human populations has resulted in several significant outbreaks of alphaviral disease (12)(13)(14). Perhaps most notable is the ongoing reemergence of chikungunya virus, which caused significant morbidity during the height of the 2006 epidemic, with as many as 40,000 new cases per week (13).Despite the range of diseases and morbidity associated within the genus, the underlying molecular life cycles are highly similar in the two hosts. Since alphaviruses are positive-sense RNA viruses, they function similarly to cellular mRNAs, relying on the translation of the incoming viral genome to ini...

The positive-sense transcripts of Sindbis virus (SINV) resemble cellular mRNAs in that they possess a 5 cap and a 3 poly(A) tail. It is likely, therefore, that SINV RNAs must successfully overcome the cytoplasmic mRNA decay machinery of the cell in order to establish an efficient, productive infection. In this study, we have taken advantage of a temperature-sensitive polymerase to shut off viral transcription, and we demonstrate that SINV RNAs are subject to decay during a viral infection in both C6/36 (Aedes albopictus) and baby hamster kidney cells. Interestingly, in contrast to most cellular mRNAs, the decay of SINV RNAs was not initiated by poly(A) tail shortening in either cell line except when most of the 3 untranslated region (UTR) was deleted from the virus. This block in deadenylation of viral transcripts was recapitulated in vitro using C6/36 mosquito cell cytoplasmic extracts. Two distinct regions of the 319-base SINV 3 UTR, the repeat sequence elements and a U-rich domain, were shown to be responsible for mediating the repression of deadenylation of viral mRNAs. Through competition studies performed in parallel with UV cross-linking and functional assays, mosquito cell factors-including a 38-kDa protein-were implicated in the repression of deadenylation mediated by the SINV 3 UTR. This same 38-kDa protein was also implicated in mediating the repression of deadenylation by the 3 UTR of another alphavirus, Venezuelan equine encephalitis virus. In summary, these data provide clear evidence that SINV transcripts do indeed interface with the cellular mRNA decay machinery during an infection and that the virus has evolved a way to avoid the major deadenylation-dependent pathway of mRNA decay.The Alphavirus genus of the Togaviridae family consists primarily of a group of viruses with single-stranded, positive-sense RNA genomes that are transmitted by arthropods and replicate exclusively within the cytoplasm of infected cells (45, 51). Sindbis virus (SINV), the prototypic virus of the genus, encodes genomic and subgenomic RNAs that are capped on the 5Ј terminus, polyadenylated at the 3Ј end, and contain 5Ј and 3Ј untranslated regions (UTRs) similar to cellular mRNAs. The viral genomic RNA, in fact, functions as an mRNA immediately upon infection. This strategy of mimicking cellular mRNAs has potential advantages and disadvantages for the virus. While this strategy allows viral RNAs to be effectively translated, viral transcripts may also be fully subject to the activity of the mRNA decay machinery within the cytoplasm of the host cell. Aspects of this anticipated interplay between SINV transcripts and cellular mRNA decay enzymes were investigated in this study.

Arthropod-borne viruses, such as the members of genus , are a significant concern to global public health. As obligate intracellular pathogens, RNA viruses must interact with the host cell machinery to establish, and complete, their viral lifecycles. Despite considerable efforts to define the host/pathogen interactions essential for alphaviral replication, an unbiased and inclusive assessment of alphaviral RNA:protein interactions has not been undertaken. Moreover, the biological and molecular importance of these interactions, in the full context of their molecular function as RNA-binding proteins, has not been fully realized. The data presented here introduces a robust viral RNA:protein discovery method to elucidate the Sindbis virus (SINV) RNA:Protein host interface. Cross-Link Assisted mRNP Purification (CLAMP) assessment reveals an extensive array of host/pathogen interactions centered on the viral RNAs (vRNAs). After prioritization of the host proteins associated with the vRNAs, we identified the site of Protein:vRNA interaction via a CLIP-seq approach and assessed the consequences of the RNA:protein binding event of hnRNP K, hnRNP I, and hnRNP M in regards to viral infection. Herein we demonstrate that mutation of the prioritized hnRNP:vRNA interaction sites effectively disrupted the hnRNP:vRNA interaction. Correlating with disrupted hnRNP:vRNA binding, SINV growth kinetics were reduced relative to wild type parental viral infections in a vertebrate and invertebrate tissue culture models of infection. The molecular mechanism leading to reduced viral growth kinetics were found to be dysregulated structural gene expression. Collectively, this study further defines the scope and importance of the alphavirus host/pathogen vRNA:protein interactions. Members of the genus Alphavirus are widely recognized for their potential to cause severe disease. Despite this recognition, there are no antiviral therapeutics, or safe and effective vaccines, currently available to treat alphaviral infection. Alphaviruses utilize the host cell machinery to efficiently establish and complete their viral lifecycle. However, the extent, and importance, of host/pathogen RNA:protein interactions is woefully under characterized. The efforts detailed in this study fulfill this critical gap; and the significance of this research is three-fold. First, the data presented here fundamentally expands the scope and understanding of alphavirus host/pathogen interactions. Secondly, this study identifies the site of interactions for several prioritized interactions and defines the contribution of the RNA:protein interaction at the molecular level. Finally, these studies build a strategy by which the importance of given host/pathogen interactions may be assessed, in the future, using a mouse model of infection.

Background: Sindbis virus RNAs bind the cellular HuR protein and cause its relocalization to the cytoplasm. Results: HuR relocalization occurs with other alphaviruses but not with several unrelated RNA viruses. It is associated with altered protein phosphorylation. Conclusion: HuR relocalization is alphavirus-selective and appears to be distinct from other types of HuR shuttling. Significance: This has potential therapeutic and diagnostic implications for alphavirus infections.

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The genus Alphavirus consists of a group of enveloped, single-stranded RNA viruses, many of which are transmitted by arthropods to a wide range of vertebrate host species. Here we report that Sindbis virus (SINV) produced from a representative mammalian cell line consists of at least two unique particle subpopulations, separable on the basis of virion density. In contrast, mosquito-derived SINV consists of a homogeneous population of particles. Our findings indicate that the denser particle subpopulation, SINV Heavy , is more infectious on a per-particle basis than SINV Light . SINV produced in mosquito cell lines (SINV C6/36 ) exhibited particle-to-PFU ratios similar to those observed for SINV Heavy . In mammalian cells, viral RNA was synthesized and accumulated more rapidly following infection with SINV Heavy or SINV C6/36 than following infection with SINV Light , due partly to enhanced translation of viral genomic RNA early in infection. Analysis of the individual particle subpopulations indicated that SINV Heavy and SINV C6/36 contain host-derived factors whose presence correlates with the enhanced translation, RNA synthesis, and infectivity observed for these particles. Members of the genus Alphavirus, of the family Togaviridae, are a group of enveloped positive-sense RNA viruses with a wide host range. For the mosquito-borne species, the virus is maintained in the enzootic cycle through transmission between a sylvatic reservoir and the mosquito host (1). The maintenance of this cycle directly affects the genetic fitness of the mosquito-borne alphaviruses. Prolonged disruption of this cycle leads to deleterious effects on viral transmission as the virus becomes adapted to a single host (2-5). Spillover from the enzootic cycle often results in the tangential infection of both humans and equines, which can result in significant outbreaks of disease. The outcome of alphaviral infection is dependent on the host system (6-12). Infection of mosquito cells does not result in the shutoff of host macromolecular synthesis and often culminates in persistent infection for the majority of mosquito cell lines (12-15). Nevertheless, cell death as a result of infection has been reported for several members of the genus in whole mosquitoes (16)(17)(18)(19)(20)(21). In contrast, infection of mammalian cells induces the shutoff of host macromolecular synthesis, resulting in a predominantly cytolytic infection. In vertebrates, the immune response to infection generally results in virus clearance. This is initiated by the recognition of viral doublestranded RNA and a rapid type I interferon (IFN-␣/␤) response (22)(23)(24)(25).Previously, we reported that the infectivity of Sindbis virus (SINV), as measured by the ratio of particles to infectious units, depends on the host cell line from which it is derived (26). SINV derived from mammalian cell lines exhibited a higher particle-to-PFU ratio, on average, than SINV generated from mosquito cell lines. This was due largely to differences in the quantity of total virus particles pr...